Download Actin microfilaments are associated with the migrating nucleus and

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Journal of Cell Science 107, 1929-1934 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
Actin microfilaments are associated with the migrating nucleus and the cell
cortex in the green alga Micrasterias
Studies on living cells
Ursula Meindl1,*, Dahong Zhang2 and Peter K. Hepler3
1Institut für Pflanzenphysiologie, Universität Salzburg, A-5020 Salzburg, Austria
2Department of Zoology, Duke University, Durham, NC 27706, USA
3Department of Biology, University of Massachusetts, Amherst, MA 01003, USA
*Author for correspondence
SUMMARY
Rhodamine-phalloidin or FITC-phalloidin has been
injected in small amounts into living, developing cells of
Micrasterias denticulata and the stained microfilaments
visualized by confocal laser scanning microscopy. The
results reveal that two different actin filament systems are
present in a growing cell: a cortical actin network that
covers the inner surface of the cell and is extended far into
the tips of the lobes in both the growing and the nongrowing semicell; it is also associated with the surface of
the chloroplast. The second actin system ensheathes the
nucleus at the isthmus-facing side during nuclear
migration. Its arrangement corresponds to that of the
microtubule system that has been described in earlier
electron microscopic investigations. The spatial correspondence between the distribution of actin filaments and
microtubules suggests a cooperation between both
cytoskeleton elements in generating the motive force for
nuclear migration. The function of the cortical actin
network is not yet clear. It may be involved in processes
like transport and fusion of secretory vesicles and may also
function in shaping and anchoring the chloroplast.
INTRODUCTION
cells that have been injected with fluorescently labeled phalloidin and analyzed by confocal laser scanning microscopy
(CLSM). Although phalloidin is a poison, when injected at low
levels it does not inhibit cell growth, or development, nor does
it block cytoplasmic streaming, yet it still stains the MFs and
allows these structures and their dynamic transformation to be
directly observed in living cells (Cleary et al., 1992; Hepler et
al., 1993; Zhang et al., 1993). The results from the present
study reveal extensive arrays of actin MFs in the cell cortex
and in the form of a cage surrounding the migrating nucleus.
Actin microfilaments (MFs) have been associated with
polarized tip growth in a variety of plant cells, especially in
fungal hyphae and pollen tubes (for references see Heath,
1990). In the desmid Micrasterias, a highly sculptured shape
emerges during cell development that consists of numerous
growing points positioned in a symmetrical pattern. Here the
participation of MFs in the regulation of development derives
mainly from studies using the drug cytochalasin (Tippit and
Pickett-Heaps, 1974; Noguchi and Ueda, 1981; Lehtonen,
1983). Culture in low concentrations of cytochalasin B, for
example, inhibits or retards cytoplasmic streaming and leads
to distinctly malformed cells. In addition, nuclear migration in
both Micrasterias and the closely related desmid, Euastrum, is
altered from the normal condition, although in this regard the
effect of cytochalasin is less profound than that caused by
microtubule inhibitors (Meindl, 1990; Url et al., 1992). Despite
these experimental inferences that MFs are involved in several
vital developmental processes, with the exception of two
reports (Noguchi and Ueda, 1988; Ueda and Noguchi, 1988),
we know remarkably little about the structural distribution of
actin in desmids. In the present investigation the spatial localization of MFs in Micrasterias has been examined in living
Key words: actin microfilament, confocal laser scanning microscopy,
Micrasterias, microinjection, nuclear migration, phalloidin
MATERIAL AND METHODS
Culture
Cells of Micrasterias denticulata Breb. were cultivated under semisterile conditions in a ‘desmid-medium’ with diluted soil extract. The
culture flasks were kept at a temperature of 20°C and a light-dark
regime of 14 to 10 hours (for detailed method see Kiermayer, 1980;
Schlösser, 1982; Meindl, 1990).
Preparation for microinjection
A small drop of nutrient solution with different developmental stages
of Micrasterias denticulata was transferred to a coverslip, which was
mounted at the bottom of a small glass chamber. A small amount of
1930 U. Meindl, D. Zhang and P. K. Hepler
warm (32-35°C), low temperature gelling agarose (type VII, Sigma)
at a concentration of 3% was placed besides the cells. The nutrient
solution containing the cells was then mixed with the agarose solution,
and the mixture was gently spread over the cover slip and immediately chilled to gel the agarose. The immobilized cells were flooded
with nutrient solution. This preparation method enabled the cells to
continue their development without any visible change.
Microinjection of phalloidin
Either FITC- or rhodamine-phalloidin (Molecular Probes Inc.) was
used for the microinjection experiments. A stock solution (3.3 µM in
methanol) was evaporated almost to dryness with nitrogen gas and
then redissolved in 100 mM KCl. The solution, which is never kept
longer than 24 hours, was sonicated and centrifuged immediately
before use. Microneedles (WPI; Kwik-fil, 1.0 mm OD) were pulled
from glass capillaries with a vertical pipette puller (David Kopf
Instruments). They were loaded with the phalloidin solution using a
thinly drawn plastic tuberculin syringe. The needle was mounted in a
Zeiss microneedle holder, connected to a Gilmont micrometer syringe
by a water-filled tubing and was maneuvered by a Narishige micromanipulator. Hydraulic pressure was applied to drive the phalloidin
solution into the cell. Microinjection was carried out on an inverted
microscope (Zeiss) equipped with Normaski optics (for further details
of the method see Zhang et al., 1990).
Microinjection impalements were made on growing semicells of
Micrasterias, preferentially into one of the growing lobes where the
cell wall was as thin as possible. This ensured that the wound caused
by a shallow injection was minimal. After injection the needle was
removed from the cell within 20 to 30 minutes thus allowing the cell
to form a plug that healed the wound and guaranteed that no
cytoplasm was lost.
Confocal laser scanning microscopy
Cells injected with phalloidin were examined on a confocal laser
scanning microscope (MRC-600, Bio-Rad) with an argon ion laser for
up to 2 hours. Observation started immediately after injection when
the needle was still in the cell. To prevent damage by the laser the
cells were exposed to irradiation at intervals of about 15 minutes for
only a few seconds. During this time pictures were taken at different
focal planes. Since it was difficult to go back to one particular focal
plane after a 15 minute development of this large cell, most emphasis
rested on showing the three dimensional MF arrangement rather than
to follow the minimal changes that occur in the orientation of the
single MFs. Thin optical sections (1-2 µm) where scanned 5 to 10
times (Kalman averaging) to form one image. When FITC-phalloidin
was used to label the cells, a red supressor filter was introduced to
reduce the autofluorescence of the chloroplast. Pictures where taken
directly from the screen after background subtraction and contrast
enhancement using either a Kodak T-Max 400 film or an Ilford Pan
F film.
RESULTS
Microinjection of either FITC- or rhodamine-phalloidin into
different developmental stages of Micrasterias denticulata
results in the appearance of two different patterns of actin MFs
a few minutes after injection. Independent from the stage of
development a network of actin MFs is present in the cortical
cytoplasmic layer directly beneath the plasma membrane (Figs
1A-C, 5A,B). This network covers the entire inner surface of
the cell including both the growing and the non-growing
semicell and also ensheathes the chloroplast (Fig. 2A-E). It
reaches far into the tips of the lobes and continues through the
isthmus area. There is no preferential orientation of the microfilament bundles and no visible correlation to the cell pattern.
When cell development continues the arrangement of the
single filaments changes but their net-like distribution is maintained.
A second fluorescent pattern that becomes visible after
microinjection of phalloidin is situated around the nucleus. In
young developmental stages only the nuclear membrane is
stained. As soon as the nucleus starts to leave the isthmus area
and migrates into the growing semicell bundles of microfilaments become visible surrounding the isthmus-facing half of
the nucleus (Fig. 3A,B). They seem to arise from a knob-like
structure that is situated exactly in the center of the isthmus
area and exhibits bright fluorescence after rhodamine-phalloidin injection (Fig. 3A). Later in cell development when the
nucleus has already moved away from the isthmus the actin
cables surrounding the nucleus merge into one thick MF
bundle running towards the isthmus and ending exactly in its
center (Fig. 4E). From optical serial sections through the area
of the nucleus it becomes obvious that the MFs construct a cage
or basket around the isthmus-facing half of the nucleus (Fig.
4A-H). When the nucleus moves back to the cell center, the
actin MF system seems to be pushed into the non-growing
semicell with the microfilament bundles passing the isthmus
area and reaching far into the non-growing semicell (Fig.
5A,B). Before the actin MFs vanish (some time after nuclear
Fig. 1. (A-C) Different optical planes of a developing semicell of Micrasterias denticulata towards the end of morphogenesis. The cell was
injected with rhodamine-phalloidin. A cortical actin network covers the inner surface of the cell and reaches far into the growing tips. A slight
background fluorescence results from the autofluorescence of the chloroplast.
Actin microfilaments in living Micrasterias cells 1931
Fig. 2. (A-E) Sequence of optical sections through a developing cell of Micrasterias denticulata injected with FITC-phalloidin. A cortical
network of actin is visible in the non-growing semicell (right) and in parts of the growing semicell. A dense actin-network also covers the
surface of the chloroplast (E).
Fig. 3. (A,B) Young bulb of Micrasterias denticulata after injection
of rhodamine-phalloidin (two different optical sections). A bundle of
actin filaments seems to arise from a bright knob-like structure and
surrounds the isthmus-facing half of the nucleus. gsc, growing
semicell.
migration is finished) their number decreases and the bundle,
which reaches into the non-growing semicell, frequently
becomes bent (Fig. 6A,B). During the entire course of nuclear
migration the nuclear envelope exhibits bright fluorescence.
DISCUSSION
By microinjection of small amounts of phalloidin into living
and growing cells two major sytems of actin MFs have been
revealed in the desmid Micrasterias. The first is an extensive
array of cortical elements, and the second consists of a cage of
clustered MFs that encircle the nucleus and appear to be
involved in nuclear migration. Although bundles of cortical
MFs similar in thickness to those reported herein had been
noted previously in fixed cells of Micrasterias examined by
fluorescence and electron microscopy (Ueda and Noguchi,
1988; Noguchi and Ueda, 1988), the prominent nucleus-associated cage has not been reported heretofore. Of added importance is the realization that the current observations have been
made on living cells, vitally stained with fluorescent phalloidin, which were continuing their development while under
microscopic examination. As in dividing stamen hair cells,
where this method has been used successfully to denote the
dynamics of actin MFs (Cleary et al., 1992, Zhang et al., 1993),
we assert that the MFs visualized in Micrasterias, under conditions in which the cells continue to grow and develop,
represent structures that bear a close and meaningful relationship to those in the unperturbed cell. We fully recognize that
phalloidin is not an ideal probe and that artifacts cannot be
completely excluded; however, the presence of normal growth
and development, when compared to control injections,
indicates in all likelihood that the actin MFs have not been
extensively bundled, stabilized or structurally rearranged.
In trying to understand the mechanism of polarized development, the current observations do not provide clear evidence
that actin MFs are involved. Not only is the pattern of phalloidin-positive material random and quite unrelated to cell
1932 U. Meindl, D. Zhang and P. K. Hepler
Fig. 4. (A-H) Different optical sections through the nuclear region of a developing cell of Micrasterias denticulata. The migrating nucleus (N)
is ensheathed by a basket-like arrangement of actin visualized after rhodamine-phalloidin injection. The nucleus is situated within the growing
semicell. An actin bundle (E,F) reaches towards the center of the isthmus area (the latter is indicated by arrows). The bright fluorescence at the
right upper area of the cell results from a bulk of chloroplast material.
Actin microfilaments in living Micrasterias cells 1933
Fig. 5. (A,B) Two different optical levels of a developing Micrasterias cell after injection of FITC phalloidin. A cortical actin network is
visible in the growing and the non-growing semicell. The nucleus (N), which is on its way back to the isthmus, is surrounded by actin bundles
reaching far into the non-growing semicell. The nuclear membrane exhibits bright fluorescence.
Fig. 6. (A,B) Same cell as in Fig. 4. but about 60 minutes later. The nucleus (N) is located close to the isthmus (isthmus indicated by arrows).
Only remnants of the actin system that originally surrounded the nucleus (compare Fig. 4) are present. The actin bundle that reaches into the
non-growing semicell is bent.
shape, but in addition we fail to see stained filaments at the
lobe tips where it would be anticipated that some form of
growth control must be exerted (Meindl, 1993). Furthermore,
the observed MFs are equally distributed between the growing
and non-growing semicell, providing additional support
against their role in polarized growth, but supporting an
involvement in cytoplasmic streaming and chloroplast
anchoring. Despite the lack of a visible and specific structural
relationship between actin MFs and polarized growth it would
be imprudent to suggest that these cytoskeletal elements were
not involved. For example, cytochalasin at levels that permit
cytoplasmic streaming, has a profound modulating effect on
cell shape development (Tippit and Pickett-Heaps, 1974;
Noguchi and Ueda, 1981; Lehtonen, 1983). Cytochalasin also
disrupts vesicle production and arrangement in the closely
related organism, Euastrum (Url et al., 1993). Furthermore, we
should recognize that phalloidin might not stain all the actin
MFs within the cell; for example, some may be closely linked
to membranes in such a way that their phalloidin-binding sites
are blocked. We argue therefore that it would be premature to
discount actin as a prime cytomorphogenetic factor; further
experimentation with other probes such as a fluorescent actin
analogue might help resolve the role of actin in polarized
growth.
The cage of actin MFs that surrounds the nucleus is an interesting new observation. Together with the microtubules, which
have been well documented in previous electron microscopic
studies (Meindl, 1983, 1992), the actin MFs are thought to contribute to the positioning and migration of the nucleus. In
attempting to decipher the relative roles of the two cytoskeletal elements it appears, however, that the MTs are more
important since their specific destruction with APM or
colchicine causes the nucleus to completely drift away from
the isthmus (Meindl and Kiermayer, 1981; Meindl, 1983),
whereas treatment with cytochalasin generates only a minor
disorientation of nuclear motion (Meindl, 1990). Not only are
MFs and MTs co-localized around the nucleus, in addition the
position of a brightly fluorescing knob, which serves as the
origin of the perinuclear actin system, corresponds exactly to
the position of the MT center visualized in earlier studies
(Meindl, 1983). Taken together these observations further
indicate that MTs and MFs cooperate in regulating the position
1934 U. Meindl, D. Zhang and P. K. Hepler
and motion of the nucleus. In conclusion, the observations
from living cells injected with phalloidin provide novel information about the localization of actin MFs and help us understand vital processes essential to the growth and development
of Micrasterias.
Parts of this study were carried out at the Plant Cell Biology Group,
Research School of Biological Sciences, Australian National University, Canberra. We are indebted to Professor Brian Gunning for many
helpful discussions and for generously providing his laboratory
equipment. We also thank Dr Ann Cleary and Dr Geoffrey Wasteneys
and other colleagues of the Plant Cell Biology Group for technical
suggestions and valuable comments. This work was supported by the
Austrian Fonds zur Förderung der wissenschaftlichen Forschung,
project 7972 to U.M., and by the US National Science Foundation,
grant #s DCB-90-04191 and DCB 93-04953 to P.K.H., and grant #
BBS-87-14235 to the Central Microscopy Facilities, University of
Massachusetts, Amherst.
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